The present invention is directed to a method of screening candidate compounds for anti-seizure activity, and which is concluded to be predictive of other adenosine releasing agent (ARA) therapeutic effects, such as anti-ischemic properties.
Procedures utilizing laboratory animals are set forth which have been found useful in screening candidate compounds for possible therapeutic potential. More specifically, described and claimed herein are procedures using agents which induce seizures in laboratory animals to screen and evaluate compounds for potential activity in preventing seizures in humans, and, thus, for usefulness as therapies for ischemic syndrome, and for seizure and related disorders and as anticonvulsant therapeutic agents.
The commonly-assigned, concurrently-filed and co-pending patent application "AICA Riboside Prodrugs", discloses the effect of certain adenosinergic agents in preventing induced seizures.
Previously, agents such as pentylenetetrazol (PTZ) have been used to induce seizures in convulsant systems which are used as models for anti-seizure activity. However, these systems are not sufficiently sensitive in measuring the anticonvulsive activity of compounds which are active in target seizure foci (i.e. areas of ATP depletion) but which have little or no effect on normal tissue.
During seizures certain neural cells fire abnormally. ATP catabolism is greatly accelerated in the abnormally firing cells leading to increased adenosine production. Adenosine has marked anticonvulsant effects and, thus, has been termed the brain's natural anticonvulsant. It appears to play a major role in the brain as an inhibitory neuromodulator; this action of adenosine is apparently mediated by certain ectocellular receptors. Adenosine has both post-synaptic and pre-synaptic effects. Among the documented effects of adenosine on nervous tissue are the inhibition of neural firing and of calcium dependent neurotransmitter release. Behaviorally, adenosine and its metabolically stable analogs have profound anticonvulsant and sedative effects.
As stated above, adenosine has been proposed to serve as a natural anticonvulsant with agents that alter its extracellular level acting as a modulator of seizure activity. Besides acting as a neuromodulator, adenosine is a potent vasodilator, an inhibitor of granulocyte oxygen free radical production, an antiarrhythmic. In fact, because of the many actions of adenosine, it has been called a "retaliatory molecule" released to protect cells against certain pathologic assaults.
Unfortunately, adenosine is toxic at concentrations that have to be administered systemically to a patient to maintain an efficacious extracellular therapeutic level at the target organ, and the administration of adenosine alone so far has been of limited therapeutic use. Likewise, since most cells in the body carry receptors for adenosine, the use of techniques that increase adenosine levels generally throughout the body can cause unwanted, dramatic changes in normal cellular physiology.
Most of the currently used antiseizure agents (including adenosine itself) exhibit side effects and toxicities or are without efficacity in many patients. There is a need for more effective anticonvulsant therapeutic compounds and strategies. An adenosinergic approach to anticonvulsant therapy appears promising. Accordingly, it is important to be able to both identify and evaluate candidate compounds for their ability to target seizure related foci in the brain without causing non-specific effects in other tissue.
Homocysteine has been shown to be a central nervous system excitant at low concentrations and a convulsant at higher levels (Folbergrova, Neuroscience 6: 1405-1411 (1981); Wuerthele et al., Life Sci. 31:2683-2692 (1982); Dewhurst et al., J. Neurochem. 40:752-757 (1983)). Studies have shown that homocysteine at high doses causes tonic-clonic seizures. In fact, a considerable portion of homocysteineuric patients (a genetic disorder characterized by cystathione B synthase deficiency resulting in elevated serum levels of homocysteine) suffer from convulsive episodes (McKusick et al., in Inherited Disorders of Sulfur Metabolism (Carson, et al. ed.) pp. 179-203 (Churchill Livingstone, London, 1971); Mudd and Levy, in The Metabolic Basis of Inherited Disease (Stanbury, et al. ed.), pp. 458-503 (McGraw Hill, New York 1978)).
Homocysteine condenses with adenosine via the enzyme S-adenosylhomocysteine hydrolase ("SAH"; E.AC.3.3.1.1.), and the convulsant action of homocysteine may be in part a result of adenosine sequestration in brain. Recent data suggest that homocysteine can sequester adenosine that is released in response to electrical stimulation (McLlwain et al. Neurochem. Int. 7:103-110 (1985)). In addition, on the basis of tissue culture data, it has been proposed that increased brain homocysteine levels can result in a decrease of brain adenosine levels and that the mechanism of homocysteine induced seizures may be related to the sequestration of adenosine.